Comparative genomics analysis and virulence-related factors in novel Aliarcobacter faecis and Aliarcobacter lanthieri species identified as potential opportunistic pathogens.

BACKGROUND: Emerging pathogenic bacteria are an increasing threat to public health. Two recently described species of the genus Aliarcobacter, A. faecis and A. lanthieri, isolated from human or livestock feces, are closely related to Aliarcobacter zoonotic pathogens (A. cryaerophilus, A. skirrowii, and A. butzleri). In this study, comparative genomics analysis was carried out to examine the virulence-related, including virulence, antibiotic, and toxin (VAT) factors in the reference strains of A. faecis and A. lanthieri that may enable them to become potentially opportunistic zoonotic pathogens. RESULTS: Our results showed that the genomes of the reference strains of both species have flagella genes (flaA, flaB, flgG, flhA, flhB, fliI, fliP, motA and cheY1) as motility and export apparatus, as well as genes encoding the Twin-arginine translocation (Tat) (tatA, tatB and tatC), type II (pulE and pulF) and III (fliF, fliN and ylqH) secretory pathways, allowing them to secrete proteins into the periplasm and host cells. Invasion and immune evasion genes (ciaB, iamA, mviN, pldA, irgA and fur2) are found in both species, while adherence genes (cadF and cj1349) are only found in A. lanthieri. Acid (clpB), heat (clpA and clpB), osmotic (mviN), and low-iron (irgA and fur2) stress resistance genes were observed in both species, although urease genes were not found in them. In addition, arcB, gyrA and gyrB were found in both species, mutations of which may mediate the resistance to quaternary ammonium compounds (QACs). Furthermore, 11 VAT genes including six virulence (cadF, ciaB, irgA, mviN, pldA, and tlyA), two antibiotic resistance [tet(O) and tet(W)] and three cytolethal distending toxin (cdtA, cdtB, and cdtC) genes were validated with the PCR assays. A. lanthieri tested positive for all 11 VAT genes. By contrast, A. faecis showed positive for ten genes except for cdtB because no PCR assay for this gene was available for this species. CONCLUSIONS: The identification of the virulence, antibiotic-resistance, and toxin genes in the genomes of A. faecis and A. lanthieri reference strains through comparative genomics analysis and PCR assays highlighted the potential zoonotic pathogenicity of these two species. However, it is necessary to extend this study to include more clinical and environmental strains to explore inter-species and strain-level genetic variations in virulence-related genes and assess their potential to be opportunistic pathogens for animals and humans.

A metabolomic study of vegetative incompatibility in Cryphonectria parasitica.

Vegetative incompatibility (VI) is a form of non-self allorecognition in filamentous fungi that restricts conspecific hyphal fusion and the formation of heterokaryons. In the chestnut pathogenic fungus, Cryphonectria parasitica, VI is controlled by six vic loci and has been of particular interest because it impedes the spread of hypoviruses and thus biocontrol strategies. We use nuclear magnetic resonance and high-resolution mass spectrometry to characterize alterations in the metabolome of C. parasitica over an eight-day time course of vic3 incompatibility. Our findings support transcriptomic data that indicated remodeling of secondary metabolite profiles occurs during vic3 -associated VI. VI-associated secondary metabolites include novel forms of calbistrin, decumbenone B, a sulfoxygenated farnesyl S-cysteine analog, lysophosphatidylcholines, and an as-yet unidentified group of lipid disaccharides. The farnesyl S-cysteine analog is structurally similar to pheromones predicted to be produced during VI and is here named 'crypheromonin'. Mass features associated with C. parasitica secondary metabolites skyrin, rugulosin and cryphonectric acid were also detected but were not VI specific. Partitioning of VI-associated secondary metabolites was observed, with crypheromonins and most calbistrins accumulating in the growth medium over time, whereas lysophosphatidylcholines, lipid disaccharide-associated mass features and other calbistrin-associated mass features peaked at distinct time points in the mycelium. Secondary metabolite biosynthetic gene clusters and potential biological roles associated with the detected secondary metabolites are discussed.

Transcriptome analysis implicates secondary metabolite production, redox reactions, and programmed cell death during allorecognition in Cryphonectria parasitica.

The underlying molecular mechanisms of programmed cell death associated with fungal allorecognition, a form of innate immunity, remain largely unknown. In this study, transcriptome analysis was used to infer mechanisms activated during barrage formation in vic3-incompatible strains of Cryphonectria parasitica, the chestnut blight fungus. Pronounced differential expression occurred in barraging strains of genes involved in mating pheromone (mf2-1, mf2-2), secondary metabolite production, detoxification (including oxidative stress), apoptosis-related, RNA interference, and HET-domain genes. Evidence for secondary metabolite production and reactive oxygen species (ROS) accumulation is supported through UPLC-HRMS analysis and cytological staining, respectively. Differential expression of mating-related genes and HET-domain genes was further examined by RT-qPCR of incompatible interactions involving each of the six vegetative incompatibility (vic) loci in C. parasitica and revealed distinct recognition process networks. We infer that vegetative incompatibility in C. parasitica activates defence reactions that involve secondary metabolism, resulting in increased toxicity of the extra- and intracellular environment. Accumulation of ROS (and other potential toxins) may result in detoxification failure and activation of apoptosis, sporulation, and the expression of associated pheromone genes. The incompatible reaction leaves abundant traces of a process-specific metabolome as conidiation is initiated.